Enterococci are among the most common bacteria isolated in nosocomial infections in the United States. Optimal antimicrobial therapy for serious enterococcal infections requires the use of synergistic combinations of a cell wall-active agent, such as ampicillin or vancomycin,with an aminoglycoside, which results in bactericidal activity against enterococci. However, many enterococcal strains have acquired aminoglycoside resistance genes that encode aminoglycoside-modifying enzymes, which eliminate this synergistic bactericidal effect. Thus, in many medical centers resistance to aminoglycosides has emerged as a major therapeutic challenge, precluding optimal therapy for the majority of enterococcal infections. High-level resistance to the clinically important aminoglycoside gentamicin in enterococci is mediated by APH(2") enzymes. We propose to study these aminoglycoside phosphotransferases in detail by (1) performing kinetics studies and investigating chemical mechanisms of these aminoglycoside-modifying enzymes; (2) investigating the roles that conserved amino acid residues play in the catalytic mechanism; and (3) studying evolution of one of these enterococcal enzymes toward higher levels and broader spectrums of aminoglycoside resistance. A clearer understanding of the interaction between these enzymes and their substrates (both aminoglycosides and the ATP cofactor), and of the evolution of the enzymes to produce a more diverse array would facilitate the development of novel aminoglycosides, either as poor substrates of the enzymes or as enzyme inhibitors, to overcome resistance conferred by these enzymes. The evolution of one of these enterococcal aminoglycoside phosphotransferases toward higher levels and broader spectrums of aminoglycoside resistance will enable predictions of which aminoglycoside antibiotics are more likely to retain their activity in the future as resistance evolves toward other aminoglycosides. Progress in either of these directions would be an important advance in the treatment of serious enterococcal infections. Effective treatment of serious infections caused by antibiotic-resistant enterococci has become a major clinical challenge. We propose to study mechanisms of resistance in enterococci to a class of antibiotics called the aminoglycosides. Results from our study will help in the development of new antibiotics and approaches to treat enterococcal infections, which are emerging rapidly as a serious threat to public health.